Sialic acid derivatives for protein derivatisation and conjugation

ABSTRACT

Derivatives are synthesized of starting materials, usually polysaccharides, having sialic acid at the reducing terminal end, in which the reducing terminal unit is transformed into an aldehyde group. Where the polysaccharide has a sialic acid unit at the non-reducing end it may be passivated, for instance by converting into hydroxyl-substituted moiety. The derivatives may be reacted with substrates, for instance containing amine or hydrazine groups, to form non-cross-linked polysialylated compounds. The substrates may, for instance, be therapeutically useful drugs peptides or proteins or drug delivery systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/568,043 having an international filing date of 12 Aug. 2004 which isthe national phase of PCT application PCT/GB2004/003511 having aninternational filing date of 12 Aug. 2004, which claims priority fromEuropean application 03254959.1 filed 12 Aug. 2003. The contents ofthese documents are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to derivatives of compounds such aspolysaccharides having at least terminal sialic units, and preferablyconsisting essentially only of sialic acid units, having an aldehydegroup for reaction with substrates at the reducing terminal end andmethods of producing them. The derivatives are useful for conversion toother reactive derivatives and for conjugation to amine-group containingsubstrates such as peptides, proteins, drugs, drug delivery systems(e.g. liposomes), viruses, cells, e.g. animal cells, microbes, syntheticpolymers etc.

Polysialic acids (PSAs) are naturally occurring unbranched polymers ofsialic acid produced by certain bacterial strains and in mammals incertain cells [Roth et. al., 1993]. They can be produced in variousdegrees of polymerisation from n=about 80 or more sialic acid residuesdown to n=2 by limited acid hydrolysis or by digestion withneuraminidases, or by fractionation of the natural, bacterially derivedforms of the polymer. The composition of different polysialic acids alsovaries such that there are homopolymeric forms i.e. the alpha-2,8-linkedpolysialic acid comprising the capsular polysaccharide of E. coli strainK1 and the group-B meningococci, which is also found on the embryonicform of the neuronal cell adhesion molecule (N-CAM). Heteropolymericforms also exist—such as the alternating alpha-2,8 alpha-2,9 polysialicacid of E. coli strain K92 and group C polysaccharides of N.meningitidis. Sialic acid may also be found in alternating copolymerswith monomers other than sialic acid such as group W135 or group Y of N.meningitidis. Polysialic acids have important biological functionsincluding the evasion of the immune and complement systems by pathogenicbacteria and the regulation of glial adhesiveness of immature neuronsduring foetal development (wherein the polymer has an anti-adhesivefunction) [Muhlenhoff et. al., 1998; Rutishauser, 1989; Troy, 1990,1992; Cho and Troy, 1994], although there are no known receptors forpolysialic acids in mammals. The alpha-2,8-linked polysialic acid of E.coli strain K1 is also known as ‘colominic acid’ and is used (in variouslengths) to exemplify the present invention.

The alpha-2,8 linked form of polysialic acid, among bacterialpolysaccharides, is uniquely non-immunogenic (eliciting neither T-cellor antibody responses in mammalian subjects, even when conjugated toimmunogenic carrier proteins) which may reflect its status as amammalian (as well as a bacterial) polymer. Shorter forms of the polymer(up to n=4) are found on cell-surface gangliosides, which are widelydistributed in the body, and are believed to effectively impose andmaintain immunological tolerance to polysialic acid. In recent years,the biological properties of polysialic acids, particularly those of thealpha-2,8 linked homopolymeric polysialic acid, have been exploited tomodify the pharmacokinetic properties of protein and low molecularweight drug molecules [Gregoriadis, 2001; Jain et. al., 2003; U.S. Pat.No. 5,846,951; WO-A-0187922]. Polysialic acid derivatisation gives riseto dramatic improvements in circulating half-life for a number oftherapeutic proteins including catalase and asparaginase [Fernandes andGregoriadis, 1996 and 1997], and also allows such proteins to be used inthe face of pre-existing antibodies raised as an undesirable (andsometimes inevitable) consequence of prior exposure to the therapeuticprotein [Fernandes and Gregoriadis, 2001]. In many respects, themodified properties of polysialylated proteins are comparable toproteins derivatised with polyethylene glycol (PEG). For example, ineach case, half-lives are increased, and proteins and peptides are morestable to proteolytic digestion, but retention of biological activityappears to be greater with PSA than with PEG [Hreczuk-Hirst et. al.,2002]. Also, there are questions about the use of PEG with therapeuticagents that have to be administered chronically, since PEG is only veryslowly biodegradable [Beranova et al., 2000] and high molecular weightforms tend to accumulate in the tissues [Bendele, et al., 1998; Conyers,et al., 1997]. PEGylated proteins have been found to generate anti PEGantibodies that could also influence the residence time of the conjugatein the blood circulation [Cheng et. al., 1990]. Despite, the establishedhistory of PEG as a parenterally administered polymer conjugated totherapeutics, a better understanding of its immunotoxicology,pharmacology and metabolism will be required [Hunter and Moghimi, 2002;Brocchini, 2003]. Likewise there are concerns about the utility of PEGin therapeutic agents that may require high dosages, since accumulationof PEG may lead to toxicity. The alpha-2,8 linked polysialic acid (PSA)therefore offers an attractive alternative to PEG, being animmunologically invisible biodegradable polymer which is naturally partof the human body, and which degrades, via tissue neuraminidases, tosialic acid, a non-toxic saccharide.

Our group has described, in previous scientific papers and in grantedpatents, the utility of natural polysialic acids in improving thepharmacokinetic properties of protein therapeutics [Gregoriadis, 2001;Fernandes and Gregoriadis; 1996, 1997, 2001; Gregoriadis et. al., 1993,1998, 2000; Hreczuk-Hirst et. al., 2002; Mital, 2004; Jain et. al.,2003, 2004; U.S. Pat. No. 5,846,951; WO-A-0187922]. Now, we describe newderivatives of PSAs, which allow new compositions and methods ofproduction of PSA-derivatised proteins (and other forms of therapeuticagent). These new materials and methods are particularly suitable forthe production of PSA-derivatised therapeutic agents intended for use inhumans and animals, where the chemical and molecular definition of drugentities is of major importance because of the safety requirements ofmedical ethics and of the regulatory authorities (e.g. FDA, EMEA).

Methods have been described previously for the attachment ofpolysaccharides to therapeutic agents such as proteins [Jennings andLugowski, 1981; U.S. Pat. No. 5,846,951; WO-A-0187922]. Some of thesemethods depend upon chemical derivatisation of the ‘non-reducing’ end ofthe polymer to create a protein-reactive aldehyde moiety (FIG. 1). Thisis because the reducing end of PSA and other polysaccharides is onlyweakly reactive with proteins under the mild conditions necessary topreserve protein conformation and the chemical integrity of PSA andprotein during conjugation. A non-reducing sialic acid terminal unit,since it contains vicinal diols, can be readily (and selectively)oxidised with periodate to yield a mono-aldehyde form, which is muchmore reactive towards proteins, and which comprises a suitably reactiveelement for the attachment of proteins via reductive amination and otherchemistries. We have described this previously in U.S. Pat. No.5,846,951 and WO-A-0187922. The reaction is illustrated in FIG. 1 inwhich

a) shows the oxidation of colominic acid (alpha-2,8 linked polysialicacid from E. coli) with sodium periodate to form a protein-reactivealdehyde at the non-reducing end and

b) shows the selective reduction of the Schiff's base with sodiumcyanoborohydride to form a stable irreversible covalent bond with theprotein amino group.

Of the various methods, which have been described to attach polysialicacids to therapeutic agents [U.S. Pat. No. 5,846,951; WO-A-0187922],none of these are specifically intended to conjugate via the reducingend, because of its weak reactivity towards therapeutic proteins.Although theoretically a useful reaction, achievement of acceptableyields of conjugate via reaction of proteins with the hemiketal of thereducing end of the PSA requires reaction times that are not conduciveto protein stability. Secondly, reactant concentrations (of polymerexcess) are required that may be unattainable or uneconomical.Nevertheless, despite the inefficiency of this reaction, we haveobserved that it gives rise to unintentional by-products duringconjugation reactions intended to produce conjugates with protein via anintroduced aldehyde at the (opposite) non-reducing end of the polymer.The potential for such by-products is evident in published studies ofcatalase, insulin and asparaginase [Fernandes and Gregoriadis, 1996,1997, 2001; Jain et. al., 2003], where the hemiketal of the natural(chemically unmodified) form of the polymer gives rise to proteinconjugates at a low level of efficiency (less than 5% of proteinbecoming derivatised, see further below in the reference examples, andtable 1) during reductive amination.

The reactivity of the reducing end of colominic acid, though weaktowards protein targets, is sufficient to be troublesome in themanufacture of chemically defined conjugates of the kind likely to bepreferred by regulatory authorities for therapeutic use in man andanimals. Unlike the natural colominic acid polymer, which is weaklymonofunctional, the periodate oxidised form of PSA (having an aldehydeat one end and a hemiketal at the other) inevitably gives rise to acomplexity of products which seriously complicate the task of producinga molecularly defined and pharmaceutically acceptable conjugate (FIG.2). FIG. 2 a is a schematic diagram showing the formation of by-productsduring polysialylation (original method). FIG. 2 b is a more detailedschematic diagram showing the formation of by-products duringpolysialylation (original method), specifically

i) asymmetric dimer;

ii) linear polymer;

iii) branched polymer; and

iv) various more-complex structures.

At first sight it would seem a simple matter to purify the intendedreaction product away from the various unintended products described inFIG. 2, however, this is by no means straightforward, since thephysicochemical characteristics of some of the intended forms (sizecharge etc.) are remarkably similar, indeed almost identical, to thoseof the intended form of the product. This would frustrate attempts topurify out the intended species from the reaction mixture by techniquessuch as ion-exchange chromatography and gel-permeation chromatography(which separate on the basis of charge and size respectively), and wouldalso frustrate many other methods of purification. Now therefore we havesolved the problems by developing a new method for conjugation ofpolysaccharides having sialic acid groups at the reducing terminal toproteins, whereby the weak reactivity of the reducing end can beexploited to beneficial effect, and which avoids the product complexitydescribed in FIG. 2( b) using the established method (FIG. 1) ofreductive amination of proteins with periodate oxidised naturalcolominic acid.

Jennings and Lugowski, in U.S. Pat. No. 4,356,170, describederivatisation of bacterial polysaccharides to proteins via an activatedreducing terminal unit involving a preliminary reduction step then anoxidation step. They suggest this approach where the reducing terminalunit is N-acetyl mannosamine, glucose, glucosamine, rhamnose and ribose.

In EP-A-0454898 an amino group of a protein is bound to an aldehydegroup produced by reducing and partially oxidising the reducing terminalsugar moiety of a glycosaminoglycan. The glycosaminoglycans treated inthis way include hyaluronic acid, chondroitin sulphate, heparin, heparansulphate, and dermatan sulphate. None of these compounds has a sialicacid unit at the reducing terminal.

In the invention there is provided a new process for producing analdehyde derivative of a sialic acid compound in which a startingmaterial having a sialic acid unit at its reducing terminal is subjectedto sequential steps of

-   -   a) reduction to reductively open the ring of the reducing        terminal sialic acid unit whereby a vicinal diol group is        formed; and    -   b) selective oxidation to oxidise the vicinal diol group formed        in step a) to form an aldehyde group.

The starting material is preferably a di-, oligo- or poly-saccharidealthough the invention may have utility for other starting materials.

The starting material used in the process of the invention shouldpreferably have the sialic acid unit at the reducing terminal end joinedto the adjacent unit through its eight carbon atom. In step b) the6,7-diol group is oxidised to form an aldehyde at the carbon 7 atom.

In an alternative embodiment, where the sialic acid unit at the reducingterminal end is joined to the adjacent unit through the 9 carbon atom,in step b) a 7, 8 diol group is formed and is oxidised to form analdehyde on the 8 carbon atom.

In the process of the invention, where the starting material is a di-,oligo- or poly-saccharide, it is preferred that the starting materialhas a terminal saccharide unit at the non-reducing end which has avicinal diol group and in which the starting material is subjected to apreliminary step, prior to step a), of selective oxidation to oxidisethe vicinal diol group to an aldehyde, whereby in step a) the aldehydeis also reduced to form a hydroxy group which is not part of a vicinaldiol group. The invention is of particular utility where the terminalunit of the reducing end of the starting material is a sialic acid unit.In an alternative embodiment the starting material may have a vicininaldiol group which is retained as such at a non-reducing terminalsaccharide unit of the starting material for step a). It will not bemodified by the reduction step, but will be oxidised in the oxidationstep to form an aldehyde group. The product will be di-functional andmay have useful therapeutic activities derived from its ability tocross-link substrates by reaction at both aldehyde groups with suitablefunctional groups on the substrate.

According to a second aspect of the invention there is provided a newprocess in which a sialic acid starting material having a terminalsialic acid at a non-reducing terminal end is subjected to the followingsteps:

-   -   c) a selective oxidation step to oxidise the non-reducing        terminal sialic acid unit at the 7, 8 vicinal diol group to form        a 7-aldehyde; and    -   d) a reduction step to reduce the 7-aldehyde group to the        corresponding alcohol. This aspect of the invention provides        sialic acid derivatives which have a passivated non-reducing        terminal, allowing activation of the reducing terminal for        subsequent reaction. The activation may be a reduction/oxidation        process e.g. of the first aspect of the invention, with optional        subsequent steps of converting the aldehyde group into another        group, such as amination to form an amine. Other steps for        activating the reducing terminal may be devised.

Preferably this second aspect of the invention is part of a process inwhich the starting material has a reducing terminal unit and is requiredto be subsequently conjugated to another molecule through that unit. Insuch a process the reducing terminal unit is generally activated forinstance by a reaction which would otherwise have activated a proportionof the sialic acid non-reducing terminal units were it not for thepassivation process. Such a reaction is, for instance selectiveoxidation of a vicinal diol moiety and is carried out after step d).

In the invention the preferred polysaccharide starting material maycomprise units other than sialic acid in the molecule. For instancesialic acid units may alternate with other saccharide units. Preferably,however, the polysaccharide consists substantially only of units ofsialic acid. Preferably these are joined 2→8 and/or 2→9.

Preferably the polysaccharide starting material has at least 2, morepreferably at least 5, more preferably at least 10, for instance atleast 50, saccharide units. For instance a polysaccharide may compriseat least 5 sialic acid units.

The polysialic acid may be derived from any source preferably a naturalsource such as a bacterial source, e.g. E. coli K1 or K92, group Bmeningococci, or even cow's milk or N-CAM the sialic acid polymer may bea heteropolymeric polymer such as group 135 or group V of N.meningitidis. The polysialic acid may be in the form of a salt or thefree acid. It may be in a hydrolysed form, such that the molecularweight has been reduced following recovery from a bacterial source. Thepolysialic acid may be material having a wide spread of molecularweights such as having a polydispersity of more than 1.3, for instanceas much as 2 or more. Preferably the polydispersity of molecular weightis less than 1.2, for instance as low as 1.01.

A population of polysialic acids having a wide molecular weightdistribution may be fractionated into fractions with lowerpolydispersities, i.e. into fractions with differing average molecularweights. Fractionation is preferably anion exchange chromatography,using for elution a suitable basic buffer. We have found a suitableanion exchange medium i) a preparative medium such as a strongion-exchange material based on activated agarose, having quaternaryammonium ion pendant groups (ie strong base). The elution buffer isnon-reactive and is preferably volatile so that the desired product maybe recovered from the base in each fraction by evaporation. Suitableexamples are amines, such as triethanolamine. Recovery may be byfreeze-drying for instance. The fractionation method is suitable for apolysialic acid starting material as well as to the derivatives. Thetechnique may thus be applied before or after the essential processsteps of this invention.

It is believed this is the first time ion-exchange chromatography hasbeen applied to fractionate an ionic polysaccharides with molecularweights above about 5 kDa especially polysialic acid of such MWs on thebasis of molecular weight. According to a further aspect of thisinvention there is provided a process for fractionating a population ofionisable polysaccharide with MW higher than 5 kDa using ion-exchangechromatography using in the elution buffer a base or acid which ispreferably volatile. Preferably the polysaccharide has carboxylic acidgroups and the ion-exchange is anion exchange. Preferably the elutionbuffer contains an amine, more preferably triethanolamine. Mostpreferably the polysaccharides are recovered from the fractions byfreeze-drying. This method can be applied for the fractionation of CAhaving other reactive moieties (maleimide or iodoacetate etc.) and othernatural (e.g. dextran sulphate) and synthetic (e.g. polyglutamic acid;polylysine in the later case by cation exchange chromatography) chargedpolymers. It is believed that it is also the first time that IEC hasbeen used to separate ionic polysaccharides in combination withprecipitation techniques and/or ultrafiltration methods. The IEC methodshould remove by-products of production which remain in the commerciallyavailable PSAs and CAs, such as endotoxins.

In a preliminary oxidation step and step c) the selective oxidationshould preferably be carried out under conditions such that there issubstantially no mid-chain cleavage of the backbone of a long-chain(polymeric) starting material, that is substantially no molecular weightreduction. Enzymes which are capable of carrying out this step may beused. Most conveniently the oxidation is a chemical oxidation. Thereaction may be carried out with immobilised reagents such aspolymer-based perrhuthenate. The most straight forward method is carriedout with dissolved reagents. The oxidant is suitably perrhuthenate, or,preferably, periodate. Oxidation may be carried out with periodate at aconcentration in the range 1 mM to 1M, at a pH in the range 3 to 10, atemperature in the range 0 to 60° C. for a time in the range 1 min to 48hours.

In the process, step a) is a step in which the sialic acid unit at thereducing end is reduced. Usually the unit at the reducing end of thestarting material is in the form of a ketal ring and reduction in stepa) opens the ring and reduces the ketone to an alcohol. The hydroxylgroup at the 6-carbon atom is thus part of a vicinal diol moiety.

Suitable reduction conditions (for steps a) and d)) may utilise hydrogenwith catalysts or, preferably hydrides, such as borohydrides. These maybe immobilised such as Amberlite (trade mark)-supported borohydride.Preferably alkali metal hydrides such as sodium borohydride is used asthe reducing agent, at a concentration in the range 1 μM to 0.1M, a pHin the range 6.5 to 10, a temperature in the range 0 to 60° C. and aperiod in the range 1 min to 48 hours. The reaction conditions areselected such that pendant carboxyl groups on the starting material arenot reduced. Where a preliminary oxidation step has been carried out,the aldehyde group generated is reduced to an alcohol group not part ofa vicinal diol group. Other suitable reducing agents arecyanoborohydride under acidic conditions, e.g. polymer supportedcyanoborohydride or alkali metal cyanoborohydride, L-ascorbic acid,sodium metabisulphite, L-selectride, triacetoxyborohydride etc.

Between any preliminary oxidation step and reduction step a) and afterstep b) and between oxidation step c) and reduction step d) and betweenstep d) and any subsequent oxidation step, the respective intermediatemust be isolated from oxidising and reducing agents, respectively, priorto being subjected to the subsequent step. Where the steps are carriedout in solution phase, isolation may be by conventional techniques suchas expending excess oxidising agent using ethylene glycol, dialysis ofthe polysaccharide and ultrafiltration to concentrate the aqueoussolution. The product mixture from the reduction step again may beseparated by dialysis and ultrafiltration. It may be possible to devisereactions carried out on immobilised oxidising and reducing reagentsrendering isolation of product straightforward.

The selective oxidation step, step b) is suitably carried out undersimilar conditions to the preliminary oxidation step as described above.Likewise the oxidation agent should be exhausted before recovery of theproduct using ethylene glycol. The product is subsequently recovered bysuitable means such as dialysis and ultrafiltration.

The process of the first aspect of the invention and of the preferredembodiment of the second aspect which includes a subsequent oxidationstep after step d) to activate a reducing terminal saccharide unitproduces an activated derivative having a reactive aldehyde moietyderived from the reducing terminal. The preferred process involving anoxidation, then reduction, then oxidation step produces an activatedproduct having a single reactive aldehyde moiety. If there is nopreliminary oxidation step and the starting material has a non-reducingterminal unit which has a vicinal diol group (e.g. a sialic acid), theproduct will have aldehyde groups at each terminal which may haveutility.

Aldehyde groups are suitable for conjugating to amine-group containingsubstrates or hydrazine compounds. Processes in which the activatedproduct of an oxidation step is subsequently conjugated to substratecompound form a further aspect of the invention. Preferably theconjugation reaction is as described in our earlier publicationsmentioned above, that is involving conjugation with an amine to form aSchiff base, preferably followed by reduction to form a secondary aminemoiety. The process is of particular value for derivatising proteins, ofwhich the amine group is suitably the epsilon amine group of a lysinegroup or the N-terminal amino group. The process is of particular valuefor derivatising protein or peptide therapeutically active agents, suchas cytokines, growth hormones, enzymes, hormones, antibodies orfragments. Alternatively the process may be used to derivatise drugdelivery systems, such as liposomes, for instance by reacting thealdehyde with an amine group of a liposome forming component. Other drugdelivery systems are described in our earlier case U.S. Pat. No.5,846,951. Other materials that may be derivatised include viruses,microbes, cells, including animal cells and synthetic polymers.

Alternatively the substrate may have a hydrazine group, in which casethe product is a hydrazone. This may be reduced if desired, foradditional stability, to an alkyl hydrazide.

In another preferred embodiment, oxidation step b) or a subsequentoxidation step after step d) is followed by the reaction of the or eachaldehyde group with a linker compound, comprising an amine group or ahydrazide group and another functional group suitable for selectivederivatisation of proteins or other therapeutically active compounds ordrug delivery systems. Such a linker may, for instance, comprise acompound having a functional group substituent for specific reactionwith sulfhydryl groups and a di-basic organic group joining the amine orhydrazide group and the functional group. Reaction of an aldehyde moietywith the amino or hydrazide group forms a reactive conjugate suitablefor binding to a substrate having a thiol (sulfhydryl) group. Suchconjugates are of particular value for selective and site-directedderivatisation of proteins and peptides.

The derivatisation of proteins and drug delivery systems may result inincreased half life, improved stability, reduced immunogenicity, and/orcontrol of solubility and hence bioavailability and pharmaco-kineticproperties, or may enhance solubility actives or viscosity of solutionscontaining the derivatised active.

According to the invention there is also provided a novel compound whichis an aldehyde derivative of a di-, oligo or polysaccharide comprisingsialic acid moieties, in which the terminal unit at the reducing end isa group OR in which R is selected from

—CH₂CH₂NHR¹, CH₂CH═N—NHR¹ and CH₂CH₂NHNHR¹ in which R¹ is H, C₁₋₂₄alkyl, aryl C₂₋₆ alkanoyl, or a polypeptide or a protein linked throughthe N terminal or the side chain amine group of a lysine residue, a drugdelivery system or is an organic group having a functional substituentadapted for reaction with a sulfhydryl group and, preferably theterminal moiety at the non-reducing end is passivated.

The novel compound may comprise mid-chain saccharide units between thetwo terminal units. The mid-chain units may consist only of sialic acidunits or, alternatively, may comprise other saccharide units in additionto the terminal units which are derived from sialic acid units. Thecompound may generally be formed as described above in relation to thefirst aspect of the invention.

The novel compound may be a polysialylated substrate, comprising atleast one polysialic acid (polysaccharide) group conjugated on eachmolecule of substrate, the conjugation including a secondary amine,hydrazone or alkyl hydrazide linkage via the reducing terminal of thepolysialic acid, and is substantially free of crosslinking via thenon-reducing end of the polysialic acid group to another molecule ofsubstrate. The substrate may be, for instance, a biologically activecompound, for instance a pharmaceutically active compound, especially apeptide or protein therapeutic, or a drug delivery system. Such activesare generally as described above.

The novel compound may have the general formula I

in which R is selected from

—CH₂CH₂NHR¹, CH₂CH═N—NHR¹ and CH₂CH₂NHNHR¹ in which R¹ is H, C₁₋₂₄alkyl, aryl C₂₋₆ alkanoyl, or a polypeptide or a protein linked throughthe N terminal or the γ-amine group of a lysine residue, a drug deliverysystem or is an organic group having a functional substituent adaptedfor reaction with a sulfhydryl group;

R³ and R⁴ are selected from

-   -   i) R³ is H and R⁴ is OH    -   ii) where R is CH(CH₂OH)CH₂OH or —CH₂CHO, R³ and R⁴ together are        ═O;    -   iii) where R is CH(CH₂OH)CH₂NHR¹ or —CH₂CH₂NHR¹, R³ is H and R⁴        is —NHR¹;    -   iv) where R is —CH(CH₂OH)CH₂NHNHR¹ or —CH₂CH₂NHNHR¹, R³ is H and        R⁴ is —NHNHR¹; or    -   v) —CH₂CH═N—NHR¹, R³ and R⁴ are together ═N—NHR¹;    -   Ac is acetyl    -   n is 0 or more; and    -   GlyO is a glycosyl group.

Where R is a group

the compound of the general formula I is the polysaccharide which ispolysialic acid derivative having an aldehyde group at the reducingterminal unit.

Where R is a group

CH₂CH═N—NHR¹ or CH₂CH₂NHNHR¹ the compound is a conjugate formed byreacting the aldehyde derivative of the polysialic acid with a hydrazideR¹NHNH₂. A hydrazide is preferably an acyl hydrazide (R¹ has a terminalcarbonyl group).

Where R is a group

or CH₂CH₂NHR¹, the compound is a conjugate formed by reacting thealdehyde derivative of the polysialic acid with a primary amine groupcontaining compound R¹NH₂.

R¹ may be the residue of a peptide or protein therapeutic, for instancean antibody or fragment, an enzyme or other biologically active compoundas described above. The group R¹ may comprise a linker moiety from theactive compound to the polysialic acid.

Alternatively, R¹ may be the residue of a linker reagent, for instanceto form a derivatised polysialic acid suitable for conjugating to groupsother than amine groups or hydrazides on active compounds. Examples arelinker reagents of the formula

that is a N-maleimido compound, in which R² is a dibasic organic group,for instance an arylene oligo(alkoxy)alkane or, preferably, alkanediylgroup, for instance a C₂₋₁₂-alkane diyl group.

The present invention is of most utility where the novel compound ismono-functional and is passivated at the terminal unit at thenon-reducing end. In such compounds R³ is H and R⁴ is OH. R can be anyof the meanings set out above. The glycosyl groups preferably comprisesialic acid units and more preferably consist only of such units, linked2-8 and/or 2-9, e.g. alternating 2-8/2-9, to one another.

The invention further provides compositions comprising the novelcompounds and a diluent as well as pharmaceutical compositionscomprising novel compounds in which R¹ has biological activity, and apharmaceutically acceptable excipient. Pharmaceutical compositions maybe administered orally, intravenously, intraperitoneally,intramuscularly, subcutaneously, intranasally, intradermally, topicallyor intratracheally.

There is provided in a second aspect of the invention a novel compoundwhich is the product of the process according to the second methodaspect which has the general formula

in which Ac is acetyl;

m is 0 or more;

Gly¹O is glycosyl; and

R⁵ is an organic group, preferably the reduced form of a terminalreducing saccharide unit, the oxidised derivative thereof which is analdehyde or the reaction product of such an aldehyde, which is, forinstance, an amine or a hydrazide.

Preferably R⁵ is selected from the same groups as R above.

Alternatively R⁵ is a group III joined via one of the carbons 8 or 9 to

(whereby the other of the carbons 8 or 9 is substituted with hydroxyl:

which is the product of the ring-opening reduction of a reducingterminal sialic acid.

Preferably the groups Gly¹O comprise sialic acid units, most preferablyconsist of sialic acid units. The value of m is preferably 2 or more,more preferably 5-1000, for instance 10-500, more preferably 10 to 50.

The new method is of particular value for creation of a monofunctionalpolysialic acid (PSA). It is based on an understanding of the tautomericequilibrium of the reducing end ring of PSA's for instance colominicacid (CA) which is described in FIG. 3. The reducing end sialic acidresidue of PSA spontaneously forms an open ring ketone bytautomerisation (FIG. 3). In the dynamic equilibrium between ring andlinear structures of the reducing end sialic acid residue, the ketonemoiety is present on only a subpopulation of PSA molecules at any oneinstant. As mentioned above however, it is here emphasized that thereactivity of the reducing end hemiketal is insufficient to be ofpractical utility for the attachment of PSA to proteins, which is whypreviously described methods do not employ this site on the polymer forattachment to proteins or other drugs. Thus as illustrated in FIG. 3, insolution, the terminal sialic acid residue at the reducing end ofpolysialic acid exists in a tautomeric equilibrium. The ring-open form,although in low abundance in the equilibrium is weakly reactive withprotein amine groups, and can give rise to covalent adducts withproteins in the presence of sodium cyanoborohydride.

In the preferred embodiment of the invention, in order to achieve betterdefined products of protein conjugation with PSAs, we have now created achemically modified form of polysialic acid that is monofunctional. Thenew form involves chemical modifications to both termini of the naturalpolysialic acid molecule. Unlike the original form of the reaction (FIG.1), wherein the polymer becomes conjugated predominantly in the 2 to 8orientation, with ‘reducing end’ outermost, the new form of the polymerbecomes attached exclusively in the opposite orientation.

The new preferred monofunctional form of the polysialic acid or otherpolysaccharide aldehyde derivative is more conducive to the synthesisand manufacture of a pharmaceutically acceptable product, since itavoids the considerable complexity which is otherwise inadvertentlycreated by use of polymer forms with unmodified reducing ends (FIG. 2).Production of the new form of the polymer (FIG. 4) involves, selectiveoxidation, preferably by periodate as in our previous disclosures, tointroduce an aldehyde function at the non-reducing end. Unlike the priorart illustrated in FIG. 1 however, this aldehyde moiety is thendestroyed by reduction, for instance with borohydride. At the other endof the polymer, the borohydride reduction step also simultaneously locksopen the ring structure of the reducing end, by reducing the hemiketal.This simultaneous reduction of the ketone to a hydroxyl moietyintroduces a new diol functionality which is now amenable to selectiveoxidation in the second oxidation step. When the natural polymer hasbeen (successively) oxidised with periodate, reduced with borohydride,and oxidised a second time with periodate, a new polymer form iscreated, which is truly monofunctional, having a single reactive group(an aldehyde) only at the reducing end (FIG. 3).

The protein reactivity (by reductive amination) of the variousintermediates described in the ‘double oxidation’ process of FIG. 4 isdescribed in Table 2. Notably, these data demonstrate that theintermediate ‘CAOR’ (colominic acid—a polysialic acid—oxidised/reduced),created by borohydride reduction of the periodate oxidised polymer, isinert towards protein targets, proving that both its aldehyde andhemiketal moieties have been destroyed by borohydride reduction. In asecond cycle of periodate oxidation of the ‘protein inert’ CAORintermediate, a new polysialic acid derivative is created (CAORO) thatis again reactive towards proteins (Table 2) and, moreover, is trulymonofunctional in character, having a single aldehyde group at the‘reducing end’ of the polymer, and being unreactive towards proteins atits other end. The monofunctional PSA can give rise only tosingle-orientation attachment to proteins, with the non-reducing endoutermost, and is incapable of inadvertently cross-linking proteins(FIG. 5). This new scheme of reaction (FIG. 4), known as the ‘doubleoxidation’ method elegantly avoids the need to purify away the intendedproduct from the various unintended products (described in FIG. 2),which are completely avoided in this new reaction scheme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a reaction scheme showing the prior art activation of thenon-reducing sialic acid terminal unit;

FIG. 1 b is a reaction scheme showing the prior art reductive aminationof the aldehyde moiety of the product of reaction scheme 1a using aprotein-amine moiety;

FIG. 2 a is a schematic diagram showing the potential side-reactionstaking place in the reaction of FIG. 1 b involving the reducingterminal;

FIG. 2 b represents schematically the potential by-products of the sidereactions of FIG. 2 a;

FIG. 3 is a reaction scheme showing the tautomerism between the ketaland ring-closed forms of the reducing terminal sialic acid unit of aPSA;

FIG. 4 a is a reaction scheme showing the preferred oxidation-reductionoxidation reactions of PSA;

FIG. 4 b gives suitable conditions for the steps of the scheme of FIG. 4and explains abbreviations used for the starting materials,intermediates and end products;

FIG. 5 is a schematic diagram similar to FIG. 2 b but shows the productsof the reaction of FIG. 4;

FIGS. 6 a-d show the results of the GPC analysis of the products ofexample 1;

FIG. 7 shows the SDS-PAGE results of example 2;

FIG. 8 shows the pharmacokinetics of the circulation half-life of theconjugates tested in vivo in mice in example 3;

FIG. 9 shows the IEC results for CA22.7 kDa in Reference example 2;

FIG. 10 shows the native PAGE results for CA22.7 kDa Reference Example2;

FIG. 11 shows the native PAGE results for several CA materials assupplied and fractions separated as in Reference example 2.2;

FIG. 12 shows GPC chromatograms for 3 of the fractions of CA separatedas in Reference example 2.2;

FIG. 13 shows native PAGE for two of the samples used in FIG. 12 andother CA and CAO samples as described in Reference example 2.2;

FIG. 14 shows the results of ultrafiltration of the CA 22.7 kDa asdescribed in Reference example 2.4;

FIG. 15 shows SDS PAGE for Example 5;

FIG. 16 shows SDS PAGE results for fractionated GH-CA conjugates formedas in Example 5;

FIG. 17 shows the results of Example 7; and

FIG. 18 (Table 6) shows ion exchange chromatography of CA22.7.

DETAILED DESCRIPTION OF THE INVENTION Examples

Materials

Ammonium carbonate, ethylene glycol, polyethylene glycol (8 KDa), sodiumcyanoborohydride (>98% pure), sodium meta-periodate and molecular weightmarkers were obtained from Sigma Chemical Laboratory, UK. The colominicacid used, linear α-(2→8)-linked E. coli K1 polysialic acids (22.7 kDaaverage, high polydispersity 1.34, 39 kDa p.d. 1.4; 11 kDa, p.d. 1.27)was from Camida, Ireland, radioactive iodide (Na¹²⁵I) was purchased fromAmersham, UK. Other materials included 2,4 dinitrophenyl hydrazine(Aldrich Chemical Company, UK), dialysis tubing (3.5 KDa and 10 KDa cutoff limits; Medicell International Limited, UK), Sepharose SP HiTrap,PD-10 columns (Pharmacia, UK), Tris-glycine polyacrylamide gels (4-20%and 16%), Tris-glycine sodium dodecylsulphate running buffer and loadingbuffer (Novex, UK). Deionised water was obtained from an Elgastat Option4 water purification unit (Elga Limited, UK). All reagents used were ofanalytical grade. A plate reader (Dynex Technologies, UK) was used forspectrophotometric determinations in protein or CA assays. CD 1 outbredmice (8-9 weeks old; 29-35 g body weight) were purchased from CharlesRiver (UK) and acclimatized for at least one week prior to their use

Methods

Protein and Colominic Acid Determination

Quantitative estimation of polysialic acids (as sialic acid) with theresorcinol reagent was carried out by the resorcinol method[Svennerholm, 1957] as described elsewhere [Gregoriadis et al., 1993;Fernandes and Gregoriadis, 1996, 1997]. Fab (protein) was measured bythe BCA colorimetric method.

Reference Example 1

Covalent PSA-protein conjugates generated by reductive amination withsodium cyanoborohydride using the natural form of polysialic acid(colominic acid, CA) from E. coli, via its weakly reactive reducing end.CA=colominic acid; CAO=oxidised colominic acid as in Fernandes andGregoriadis, 1996; Jain, et al., 2003. Sodium cyanoborohydride (NaCNBH₃)was used at a concentration of 4 mg ml⁻¹.

The results are shown in Table 1. The molar ratios in column 1 are theratio of starting CA(O) to protein. (n=3, ±standard deviation).

TABLE 1 Degree of modification with CA molar ratio Preparation(CA:protein) Catalase + CAO + NaCNBH₃ (10:1) 0.77 ± 0.16 Catalase +CAO + NaCNBH₃ 50:1) 2.59 ± 0.08 Catalase + CA + NaCNBH₃ (50:1) 0.55 ±0.05 Catalase + CA (50:1) 0.65 ± 0.04 Insulin + CAO + NaCNBH₃ (25:1)1.60 ± 0.14 Insulin + CAO + NaCNBH₃ (50:1) 1.65 ± 0.14 Insulin + CAO +NaCNBH₃ (100:1) 1.74 ± 0.12 Insulin + CA + NaCNBH₃ (25:1) 0.20 ± 0.02Insulin + CA + NaCNBH₃ (50:1) 0.21 ± 0.04 Insulin + CA + NaCNBH₃ (100:1)0.24 ± 0.06

Example 1 Preparation of Monofunctional Polysialic Acid

1a Activation of Colominic Acid

Freshly prepared 0.1 M sodium metaperiodate (NaIO₄) solution was mixedwith CA (100 mg CA/ml NaIO₄) at 20° C. and the reaction mixture wasstirred magnetically for 15 min in the dark. A two-fold volume ofethylene glycol was then added to the reaction mixture to expend excessNaIO₄ and the mixture left to stir at 20° C. for a further 30 min. Theoxidised colominic acid was dialysed (3.5 KDa molecular weight cut offdialysis tubing) extensively (24 h) against a 0.01% ammonium carbonatebuffer (pH 7.4) at 4° C. Ultrafiltration (over molecular weight cut off3.5 kDa) was used to concentrate the CAO solution from the dialysistubing. Following concentration to required volume, the filterate waslyophilized and stored at −40° C. until further use.

1b Reduction of Colominic Acid

Oxidised colominic acid (CAO; 22.7 kDa) was reduced in presence ofsodium borohydride. Freshly prepared 0.15 mM sodium borohydride (NaBH₄;in 0.1M NaOH diluted to pH 8-8.5 by diluting with dilute H₂SO₄ solution)was mixed with CAO (100 mg CA/ml) at 20° C. and the reaction mixture wasstirred for up to 2 h in the dark. The pH was brought down to 7 by thecompletion of the reaction. The oxidised/reduced colominic acid (CAOR)was dialysed (3.5 KDa molecular weight cut dialysis tubing) against0.01% ammonium carbonate buffer pH (7) at 4° C. Ultracentrifugation wasused to concentrate the CAOR solution from the dialysis tubing. Thefiltrate was lyophilized and stored at 4° C. until further required. Thedetermination of any aldehyde content was determined as described under‘determination of CA oxidation’.

1c Reoxidation of CA

After confirmation of no aldehyde content the oxidised/reduced colominicacid (CAOR) was again oxidised as reported under activation of colominicacid except CAOR was incubated with periodate solution for longer time(up to 1 h). The degree of oxidation in the CAORO product was measuredon lyophilized powder obtained from this stage as well.

1d Determination of the Oxidation State of CA and Derivatives

Qualitative estimation of the degree of colominic acid oxidation wascarried out with 2,4 dinitrophenylhydrazine (2,4-DNPH), which yieldssparingly soluble 2,4 dinitrophenyl-hydrazones on interaction withcarbonyl compounds. Non-oxidised (CA), oxidised (CAO), reduced (CAOR)and re-oxidised (CAORO) (5 mg each), were added to the 2,4-DNPH reagent(1.0 ml), the solutions were shaken and then allowed to stand at 31° C.until a crystalline precipitate was observed [Shriner et. al., 1980].The degree (quantitative) of CA oxidation was measured with a method[Park and Johnson, 1949] based on the reduction of ferricyanide ions inalkaline solution to ferric ferrocyanide (Persian blue), which is thenmeasured at 630 nm. In this instance, glucose was used as a standard.

1e Gel Permeation Chromatography

Colominic acid samples (CA, CAO, CAOR and CAORO) were dissolved in NaNO₃(0.2M), CH₃CN (10%; 5 mg/ml) and were chromatographed on over2×GMPW_(XL) columns with detection by refractive index (GPC system:VE1121 GPC solvent pump, VE3580 RI detector and collation with Trisec 3software (Viscotek Europe Ltd). Samples (5 mg/ml) were filtered over0.45 μm nylon membrane and run at 0.7 cm/min with 0.2M NaNO₃ and CH₃CN(10%) as the mobile phase.

Results

Colominic acid (CA), a polysialic acid, is a linear alpha-2,8-linkedhomopolymer of N-acetylneuraminic acid (Neu5Ac) residues (FIG. 1 a).Periodate, however, is a powerful oxidizing agent and although selective[Fleury and Lange, 1932] for carbohydrates containing hydroxyl groups onadjacent carbon atoms, it can cause time-dependent cleavage to theinternal Neu5Ac residues. Therefore, in the present work exposure ofcolominic acids to oxidation was limited to 15-60 min using 100 mMperiodate at room temperature [Lifely et. al., 1981]. Moreover, asperiodate decomposes on exposure to light to produce more reactivespecies [Dyer, 1956], reaction mixtures were kept in the dark. Theintegrity of the internal alpha-2,8 linked Neu5Ac residues postperiodate and borohydride treatment was analysed by gel permeationchromatography and the chromatographs obtained for the oxidised (CAO),oxidised reduced (CAOR), double oxidised (CAORO) materials were comparedwith that of native CA. It was found (FIG. 6) that oxidized (15 minutes)(CAO) (6b), reduced (CAOR) (6c), double oxidised (1 hr) (CAORO) (6d) andnative (6a) CA exhibit almost identical elution profiles, with noevidence that the successive oxidation and reduction steps give rise tosignificant fragmentation of the polymer chain. The small peaks areindicative of buffer salts.

Quantitative measurement of the oxidation state of CA was performed byferricyanide ion reduction in alkaline solution to ferrocyanide(Prussian Blue) [Park and Johnson, 1949] using glucose as a standard[results are shown in table 2]. Table 2 shows that the oxidizedcolominic acid was found to have a greater than stoichiometric (>100%)amount of reducing agent, i.e. 112 mol % of apparent aldehyde contentcomprising the combined reducing power of the reducing end hemiketal andthe introduced aldehyde (at the other end). No reactivity was seen inCAOR demonstrating that the neutralisation of both the aldehyde and thehemiketal of CAO had been successfully accomplished by borohydridereduction. After the second cycle of periodate oxidation, the aldehydecontent of the polymer was restored to 95% in CAORO (within experimentalerror of 10%) demonstrating the successful introduction of a newaldehyde moiety at the reducing end.

The results of quantitative assay of colominic acid intermediates in thedouble oxidation process using ferricyanide (Table 2) were consistentwith the results of qualitative tests performed with 2,4dinitrophenylhydrazine which gave a faint yellow precipitate with thenative CA, and intense orange colour with the aldehyde containing formsof the polymer, resulting in an intense orange precipitate after tenminutes of reaction at room temperature.

TABLE 2 Table 2: Degree of oxidation of various colominic acidintermediates in the double oxidation reaction scheme using glucose as astandard (100%, 1 mole of aldehyde per mole of glucose; n = 3 ± s.d). CAspecies Degree of oxidation colominic acid (CA)  16.1 ± 0.63 colominicacid-oxidised (CAO) 112.03 ± 4.97 colominic acid-reduced (CAOR) 0; Notdetectable colominic acid-oxidised-  95.47 ± 7.11 reduced-oxidised(CAORO)

Example 2 Preparation of Fab-Colominic Acid Conjugates

Fab was dissolved in 0.15 M PBS (pH 7.4) and covalently linked todifferent colominic acids (CA, CAO, CAOR and CAORO) via reductiveamination in the presence of sodium cyanoborohydride (NaCNBH₃).Colominic acid from each step of the synthesis (starting material andproducts of each of Examples 1a to c) together with Fab in a CA:Fabmolar ratios (100:1) were reacted in 0.15 M PBS (pH 7.4; 2 ml)containing sodium cyanoborohydride (4 mg/ml) in sealed vessels withmagnetic stirring at 35±2° C. in an oven. The mixtures was thensubjected to ammonium sulphate ((NH₄)₂SO₄) precipitation by adding thesalt slowly whilst continuously stirring, to achieve 70% w/v saturation.The samples, stirred for 1 h at 4° C., were centrifuged for 15 min(5000×g) and the pellets containing polysialylated Fab suspended in asaturated solution of (NH₄)₂SO₄ and centrifuged again for 15 min(5000×g). The precipitates recovered were redissolved in 1 ml 0.15M Naphosphate buffer supplemented with 0.9% NaCl (pH 7.4; PBS) and dialysedextensively (24 h) at 4° C. against the same PBS. The dialysates werethen assayed for sialic acid and Fab content and the conjugation yieldwas expressed in terms of CA:Fab molar ratio. Controls includedsubjecting the native protein to the conjugation procedure in thepresence of non-oxidised CA or in the absence of CA, under theconditions described. Stirring was kept to a minimum to avoidconcomitant denaturation of the protein. Polysialylated Fab was furthercharacterised by size exclusion chromatography, ion exchangechromatography and SDS-PAGE.

2b Ion Exchange Chromatography

Zero (control) and 48 h samples (0.5 ml) from the reaction mixtures weresubjected to ion exchange chromatography (IEC) on a Sepharose SP cationexchange column (1 ml; flow rate 1 ml/min; binding/washing buffer 50 mMsodium phosphate, pH 4.0; elution buffer, 50 mM sodium phosphate buffer,pH 4.0 containing 1M sodium chloride). The columns were washed, elutedand the eluent fractions were assayed for CA and protein (Fab) content.PD-10 columns were used for desalting samples before applying to column.

2c SDS-Polyacrylamide Gel Electrophoresis

SDS-PAGE (MiniGel, Vertical Gel Unit, model VGT 1, power supply modelConsort E132; VWR, UK) was employed to detect changes in the molecularsize of Fab upon polysialylation. SDS-PAGE of Fab and its conjugates(with CA, CAO, CAOR and CAORO) of 0 (control) and 48 h samples from thereaction mixtures as well as a process control (non oxidised CA), wascarried out using a 4-20% polyacrylamide gel. The samples werecalibrated against a wide range of molecular weight markers.

In previous experiments [Jain et. al., 2003; Gregoriadis, 2001] withother proteins it was found that optimal CA:Fab (derived from sheep IgG)molar conjugation yields required a temperature of 35±2° C. in 0.15 MPBS buffer at pH 6-9 for 48 h. The imine (Schiff base) species formedunder these conditions between the polymer aldehyde and protein wassuccessfully reduced with NaCNBH₃ to form a stable secondary amine[Fernandes and Gregoriadis, 1996; 1997]. Exposure of protein toperiodate-oxidised natural CA generates a metastable Schiff's baseCA-protein adduct (as reported for the polysialylation of catalase)[Fernandes and Gregoriadis, 1996]. Likewise, in the reaction of oxidisedforms of CA with Fab, we first created a metastable Schiff's baseadduct, by incubation of the oxidised polymer with Fab for 48 h at 37°C. which was then consolidated by selective reduction (reductiveamination) with NaCNBH₃ (which reduces the Schiff's base iminestructure, but not the aldehyde moiety of the polymer). In order tocharacterise the protein reactivity of the various CA intermediates ofthe ‘double oxidation method’ Fab was subjected to reductive aminationin the presence of natural CA (CA), CA oxidized (CAO), CAoxidised-reduced (CAOR) and CA ‘double oxidised’(CAORO). For thesestudies 22.7 kDa PSA was used, at CA:Fab molar ratio of (100:1). After48 h of incubation in the presence of NaCNBH₃, Fab conjugates wereisolated from reaction mixtures by precipitation with ammonium sulphate(as described in the “Examples”) and the results expressed in terms ofCA:Fab molar ratios in the resulting conjugates (Table 3).

TABLE 3 Synthesis of Fab (protein) colominic acid compounds. Molarconjugation CA species tested ratio (CA:Fab) attained colominic acid(CA) 0.21:1 (weakly reactive) colominic acid-oxidised (CAO) 2.81:1(highly reactive) colominic acid-oxidised- not detectable reduced (CAOR)(reactivity destroyed) colominic acid-oxidised- 2.50:1 reduced-oxidised(CAORO) (high reactivity regained)

It is evident from Table 3 that when natural, non-oxidized CA (in thepresence of cyanoborohydride) was used, a significant but low level ofconjugation was observed (resulting in a 0.21:1, CA:Fab molar ratio) viareaction with the hemiacetal group of CA at its reducing end.

Formation of the CA-Fab conjugates was further confirmed by theco-precipitation of the two moieties on addition of (NH₄)₂SO₄ (CA assuch does not precipitate in the presence of the salt). Evidence ofconjugation was also confirmed by ion exchange chromatography (IEC, notshown) and polyacrylamide gel electrophoresis (SDS-PAGE; FIG. 7).

For ion-exchange chromatography, polysialylated Fab obtained by(NH₄)₂SO₄ precipitation was redissolved in sodium phosphate buffer (50mM, pH 4.0) and subjected to IEC using a Sepharose SP HiTrap column(cation exchange). In contrast with results indicating completeresolution of CA (in the wash) and Fab (in eluted fractions), both CAand Fab from the 48 h reaction samples co-eluted in the wash fractions,demonstrating the presence of CA-Fab conjugate.

FIG. 7 describes the analysis of the antibody Fab conjugates describedabove. These data confirm that the molecular weight distributions of thetwo conjugates are very similar (as expected, since the byproductsobtained from the asymmetrically bifunctional CA make up only a smallpercentage of the total population of molecules). It is also evidentfrom FIG. 7 that whether Fab conjugates were prepared fromasymmetrically bifunctional CA (i.e. periodate oxidised natural CA) orfrom monofunctional PSA, that conjugates of a wide molecular weightdistribution, elevated from the molecular weight of uderivatised Fabcontrol, were created. This is consistent with the known polydispersityof the natural polymer reported in our previous published works. FIG. 7also confirms that reductive amination with monofunctional CA gives riseto an Fab conjugate with comparable yield to that of the earlier methodbased on periodate oxidised natural CA (described in FIG. 1). It is alsoevident from FIG. 7 that only trace amounts of underivatised Fabremained in each conjugate sample. The trace amounts of remaining Fabwere removed from these conjugates by ion exchange chromatography priorto in vivo studies (Example 3 below).

Example 3 In Vivo Studies

Samples of sheep IgG Fab fragment or conjugates with CAO or CAORO wereradiolabelled with I¹²⁵ as follows:

10% by volume of each of these samples was removed (˜100 μl) and placedinto fresh IODO-gen tubes. A 20 μl sample of PBS containing 200 mCi of¹²⁵I (as NaI) was added to the protein or conjugate and the tubes werecapped and allowed to incubate at room temperature for 10 min. Thecontents of the tubes were then transferred to 500 μl centrifugalfilters (3.5 kDa m. w. cut off) and the samples spun at 6,500 rpm in amicrocentrifuge. The eluent was discarded and the volume in theretentate (above the membrane) made up to 500 μl. This process wasrepeated a further 5 times after which the radioactivity above (protein)and below (free iodine) the membrane for a 5 μl sample was assessedusing a Packard Cobra Gamma counter. If the counts due to free I¹²⁵ wereless than 5% of those in the conjugated fraction, no furtherpurification was carried out. If the free I¹²⁵ was >5% the purificationcycle was repeated and the samples re-assessed.

CD1 mice (29-35 g body weight) were dosed with 40 μg (100 μl volume inPBS) of protein per mouse (˜1.6 mg/kg) by the i.v. route (tail vein) asa single injection and 50 μl samples of blood were then taken (usingheparinised graduated capillaries) at time intervals from a differenttail vein and added into 500 μl PBS. The last bleed recorded was a totalbleed in order to allow sufficient counts. Samples were then centrifugedat 3000 rpm for 10 minutes and recorded supernatant removed and placedin gamma counter tubes. Samples were counted along with representativesamples of the injected protein in a Packard Cobra II auto gammacounter. Recorded counts were expressed as a percentage of the originaldose injected.

Samples of radio-iodinated Fab, and CAO and CAORO Fab conjugates, andinjected intravenously into mice to monitor half-life in the bloodcirculation. FIG. 8 shows the pharmacokinetics of native Fab VsFab-colominic acid conjugates prepared by the original method (usingCAO) and by the new double-oxidation method (using CAORO). These resultsdemonstrate that CAO-Fab and CAORO-Fab gave rise to marked andsignificantly longer residence times in the circulation, than was thecase for underivatised Fab, giving rise to increases of 6.28 fold and5.28 fold (respectively) in AUC values compared to native Fab.

Example 4 Synthesis of Maleimide Conjugate

The CAORO synthesised in Example 1c above was reacted with 5 molarequivalents of N-[β-maleimidopropionic acid] hydrazide in 0.1M sodiumacetate for 2 h at 37° C. The product hydrazone was precipitated inethanol, resuspended in sodium acetate and precipitated again inethanol, redissolved in water and freeze-dried. The product is usefulfor site-specific conjugation to the thiol groups of cysteine moietiesin proteins and peptides.

The monofunctional polysialic acid aldehyde derivative could also bereacted with a linking compound having a hydrazide moiety and aN-maleimide moiety to form a stable hydrazone having an active maleimidefunctionality useful for reacting with a thiol group.

Reference Example 2 Fractionation of Colominic Acid by Ion ExchangeChromatography (CA, 22.7 KDa, pd 1.34) Reference Example 2.1Fractionation at Large Scale

An XK50 column (Amersham Biosciences, UK) was packed with 900 mlSepharose Q FF (Amersham Biosciences) and equilibrated with 3 columnvolumes of wash buffer (20 mM triethanolamine; pH 7.4) at a flow rate of50 ml/min. CA (25 grams in 200 ml wash buffer) was loaded on column at50 ml per minute via a syringe port. This was followed by washing thecolumn with 1.5 column volumes (1350 ml) of washing buffer.

The bound CA was eluted with 1.5 column volumes of different elutionbuffers (Triethanolamine buffer, 20 mM pH 7.4, with 0 mM to 475 mM NaClin 25 mM NaCl steps) and finally with 1000 mM NaCl in the same buffer toremove all residual CA and other residues (if any).

The samples were concentrated to 20 ml by high pressure ultra filtrationover a 5 kDa membrane (Vivascience, UK). These samples were bufferexchanged into deionised water by repeated ultra filtration at 4° C. Thesamples were analysed for average molecular weight and other parametersby GPC (as reported in example 1e) and native PAGE (stained with alcianblue).

Reference Example 2.2 Fractionation at Smaller Scale

The following samples were fractionated using an identical wash andgradient system on a smaller scale (up to 75 ml matrix; 0.2-3 gram ofcolominic acid):

Colominic acid (CA, 22.7 kDa, pd 1.34; CA, 39 KDa, pd=1.4), colominicacid-aldehyde (CAO, 22.7 kDa, pd 1.34), monofunctional colominic acid(CAORO, 22.7 kDa; pd 1.34), colominic acid-amine (CA-NH2, 22.7 kDa, pd1.34), colominic acid maleimide (CAM, as per example 4 and the m.w. ofCA produced monitored throughout).

Narrow fractions of CA produced using above procedure were oxidised with10 mM periodate and analysed by gel permeation chromatography (GPC) andnative PAGE for gross alteration to the polymer.

Results

TABLE 4 Ion exchange chromatography of CA22.7: Scale up (75 ml matrix, 3g of CA) Elution buffers (in 20 mM Triethanolamine % buffer + mM NaCl,pH 7.4) M.W. Pd Population 325 mM 12586 1.091 77.4% 350 mM 20884 1.0373.2% 375 mM 25542 1.014 5.0% 400 mM 28408 1.024 4.4% 425 mM □ □ 7.4% 450mM 43760 1.032 2.3% 475 mM 42921 1.096 0.2% *Not done

Colominic acid and its derivatives (22.7 kDa) were successfullyfractionated into various narrow species with a polydispersity less than1.1 with m.w. averages of up to 46 kDa with different % of populations.FIGS. 9 and 10; Table 4 show the results of separating the 22.7 kDamaterial at a scale of 75 ml. FIG. 9 is the GPC result and FIG. 10 is anative PAGE.

This process was scalable from 1 ml to 900 ml of matrix with thefractionation profile almost identical at each scale (not all resultsshown).

The fractionation of larger polymer (CA, 39 kDa, pd 1.4) producedspecies up to 90 kDa. This process can successfully be used for thefractionation of even large batches of the polymer. FIG. 11 shows thenative PAGE results for the 3 CA samples as supplied and for fractionsseparated by ion-exchange analysed as in Table 4. The PAGE results showthat the ion exchange fractions are narrowly dispersed. This isconsistent with the GPC data shown in FIG. 12 which shows the resultsfor 3 of the fractions separated from the 22.7 kDa CA. The retentionvolumes are shown in Table 5.

TABLE 5 Sample MW Mn PD 1 18727 15016 1.25 2 27677 25095 1.10 3 4095040279 1.02

The 22.7 kDa material is separated on a larger scale. Using GPC thefractions from ion exchange are analysed. The following fractions shownin Table 6 (see FIG. 18) were recovered.

All narrow fractions were successfully oxidised with 10 mM periodate andsamples taken from different stages of the production process andanalysed by GPC and native PAGE showed no change in the molecular weightand polydispersity The data for some of the samples are shown in FIG.13.

2.3 Precipitation of Colominic Acid

Differential ethanol precipitation was used to precipitate differentchain lengths of colominic acid.

Results

Differential ethanol precipitation showed that smaller CAs required moreethanol (EtOH). Broad p.d. 22.7 kDa polymer was precipitated with 70%EtOH giving a yield>80% of product polymer. A concentration of 80% EtOHwas required to precipitate>80% of a lower MW 6.5 KDa (pd<1.1). Thisprocess also removes any salt contaminating the product.

2.4 Fractionation of Colominic Acid by Filtration

Samples of 22.7 kDa were purified by ultrafiltration over differentmolecular weight cut off membranes (5, 10, 30, 50, and 100 kDa). In allcases retentate was examined by GPC and native PAGE.

Results

Samples of 22.7 kDa were purified by ultrafiltration over differentmolecular weight cut off membranes showed that there was a decrease inpolydispersity of the polymer and a shift towards higher molecularweight with increase in membrane cut off (FIG. 14).

Combined methods and ion pair chromatography can also be forfractionation of the polymers.

Example 5 Synthesis of Growth Hormone (GH)-Colominic Acid Conjugates(Broad and Narrow Dispersed)

Colominic acid-oxidised (CAO; 22.7 kDa)) and narrow dispersed-colominicacid-oxidised (NCAO; 27.7 kDa pd=1.09; 40.9 kDa. pd=1.02) prepared inReference example 2.2 was used for the preparation of GH conjugates.

Preparation of Growth Hormone-Colominic Acid Conjugates

Growth hormone was dissolved in 0.15 M PBS (pH 7.4) and covalentlylinked to different colominic acids (CAO and NCAO). Different CAs (22.7kDa, CAO; 27.7 kDa & 40.9 kDa, NCA)) were individually added to GH (2mg) in a CA:GH molar ratios (12.5:1), sodium cyanoborohydride was addedto a final concentration of 4 mg/ml. The reaction mixtures were sealedand stirred magnetically for 24 h at 35±2° C. The mixtures were thensubjected to ammonium sulphate ((NH₄)₂SO₄) precipitation by adding thesalt slowly whilst continuously stirring, to achieve 70% w/v saturation,stirred for 1 h at 4° C., then spun (5000×g) for 15 min and the pelletsresuspended in a saturated solution of (NH₄)₂SO₄ and spun again for 15min (5000×g). The precipitates recovered were redissolved in 1 ml PBS pH7.4 and dialysed extensively (24 h) at 4° C. against the same buffer.Controls included subjecting the native protein to the conjugationprocedure in the presence of non-oxidised CA or in the absence of CA.Shaking was kept to a minimum to avoid concomitant denaturation of theprotein. Polysialylated GH was characterised by SDS-PAGE. Thepolysialylated GH was passed through anion exchange chromatography asdescribed in Reference example 2 and the product fractions subjected toSDS PAGE.

Results

The results (FIG. 15) show that in control well (with GH) the migrationof the sample is similar to that for fresh GH. In the conjugate lanesthere are shifts in the bands which typically indicates an increase inmass indicative of a polysialylated-GH. The band width was significantlynarrowed down in case of conjugates with narrow dispersed polymer incomparison to conjugates with broad dispersed polymers. Further, GHconjugates (with broad dispersed polymer) were separated into differentspecies by anion exchange chromatography (FIG. 16).

Example 6 Synthesis of Insulin-Colominic Acid Conjugates

Activated polysialic acid (colominic acid-oxidised (CAO)) andmonofunctional polysialic acid (colominic acid-oxidised-reduced-oxidised(CAORO)) prepared in example 1 was used for the preparation ofrh-insulin conjugates.

Preparation of Insulin-Colominic Acid Conjugates

Insulin was dissolved in a minimum volume of 15 mM HCl followed bydilution with 0.15 M PBS (pH 7.4) and covalently linked to differentcolominic acids (CA, CAO and monofunctional CAORO). Colominic acid (22.7kDa) together with insulin (2 mg) in a CA:insulin molar ratios (25:1)were reacted for 48 h in 0.15 M PBS (pH 7.4; 2 ml) containing sodiumcyanoborohydride (4 mg/ml) in sealed vessels with magnetic stirring at35±2° C. in an incubator. The mixtures was then subjected to ammoniumsulphate ((NH₄)₂SO₄) precipitation by adding the salt slowly whilstcontinuously stirring, to achieve 70% w/v saturation. The samples werestirred for 1 h at 4° C., then spun (5000×g) for 15 min and the pelletssuspended in a saturated solution of (NH₄)₂SO₄ and centrifuged again for15 min (5000×g). The precipitates recovered were redissolved in 1 ml0.15M Na phosphate buffer supplemented with 0.9% NaCl (pH 7.4; PBS) anddialysed extensively (24 h) at 4° C. against the same PBS. Thedialysates were then assayed for sialic acid and protein content and theconjugation yield was expressed in terms of CA:insulin molar ratio (asper example 1). Controls included subjecting the native protein to theconjugation procedure in the presence of non-oxidised CA or in theabsence of CA, under the conditions described. Shaking was kept to aminimum to avoid concomitant denaturation of the protein. Polysialylatedinsulin was further characterised by ion exchange chromatography andSDS-PAGE. Results are expressed in terms of CA:insulin molar ratios inthe resulting conjugates (Table 7).

TABLE 7 Synthesis of insulin (protein) colominic acid compounds Molarconjugation CA species tested ratio (CA:insulin) attained colominic acid(CA) 0.07:1 (weakly reactive) colominic acid-oxidised (CAO) 1.60:1(highly reactive) colominic acid-oxidised- 1.35:1 reduced-oxidised(CAORO) (high reactivity regained) (monofunctional)

It is evident from Table 7 that when natural, non-oxidized CA (in thepresence of cyanoborohydride) was used, a significant but low level ofconjugation was observed (resulting in a 0.07:1, CA:insulin molar ratio)via reaction with the hemiacetal group of CA at its reducing end.

Formation of the CA-insulin conjugates was further confirmed by theco-precipitation of the two moieties on addition of (NH₄)₂SO₄ (CA assuch does not precipitate in the presence of the salt). Evidence ofconjugation was also confirmed by ion exchange chromatography (IEC) andpolyacrylamide gel electrophoresis (SDS-PAGE).

Example 7 In Vivo Studies

Insulin and polysialylated insulin constructs of Example 6 were testedfor their ability to reduce blood glucose level in normal female T/Ooutbred mice (22-24 gram body weight). Animals were divided into groupsof five, injected subcutaneously (s.c.) with insulin (0.3 units permouse in 0.9% sodium chloride or with the same protein equivalence ofpolysialylated insulin) and glucose levels in blood samples weremeasured at time intervals using a glucose assay kit (Accu-ChekAdvantage, Roche, UK).

Results

The pharmacological activity of polysialylated insulin constructs wascompared with that of intact insulin in normal mice injectedsubcutaneously and bled at time intervals. The blood glucose levels ofthe mice for the 3 insulins are shown in FIG. 17. The data points showthe average of 5 samples and the error bars are the s.e.m. values.Results in FIG. 17 clearly show that polysialylated insulins (preparedby original method (using CAO) and by the new double-oxidation method(using monofunctional CAORO)) exerted a more prolonged reduction ofblood glucose levels. Thus, whereas glucose levels attained nadir valuesat 0.75 hours to return to normal levels two hours after treatment withintact insulin, glucose levels in mice treated with the polysialylatedpeptide, although also lowest at 0.75 h, returned to normal values at 6hours. These results demonstrate that CAO-insulin and CAORO-insulin gaverise to marked and significantly longer residence times in thecirculation, than was the case for underivatised insulin, giving rise toincreases in area above curve compared to native insulin.

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The invention claimed is:
 1. A process for producing an aldehyde derivative of a reducing terminal sialic acid of a starting material having a sialic acid at a non-reducing terminal, which process comprises: a) a selective oxidation step to oxidise the non-reducing terminal sialic acid unit at the 7,8 vicinal diol group to form a 7-aldehyde; b) a reduction step to reduce the 7-aldehyde group of the non-reducing terminal sialic acid to a corresponding alcohol, wherein the reduction step additionally reduces the reducing terminal sialic acid to effect ring opening of the reducing terminal sialic acid whereby a vicinal diol group is formed; c) an oxidation step which oxidises the reducing terminal unit but does not oxidise the moiety produced in the oxidation and reduction reactions of the non-reducing terminal unit; and d) a conjugation step to conjugate the reducing terminal sialic acid to a substrate.
 2. A process according to claim 1 in which the starting material is a di-, oligo- or poly-saccharide.
 3. A process according to claim 2 in which the polysaccharide comprises midchain sialic acid units.
 4. A process according to claim 1 in which oxidation step a) is carried out in aqueous solution in the presence of periodate at a concentration in the range 1 μM to 1 M, a pH in the range 3 to 10, a temperature in the range 0 to 60° C. and a time in the range 1 min to 48 hours.
 5. A process according to claim 1 in which the reduction step (b) is carried out in aqueous solution in the presence of borohydride at a concentration in the range 1 μM to 0.1 M, a pH in the range 6.5 to 10, a temperature in the range 0 to 60° C. and a period in the range 1 min to 48 h.
 6. A compound of formula II

wherein Ac is acetyl; m is 0 or more; Gly¹O is glycosyl; and R⁵ is an organic group conjugated to a reducing terminal sialic acid; in which R⁵ is

and in which R¹ is a peptide, a polypeptide, a protein, a protein therapeutic, a drug, a drug delivery system, a liposome, a virus, a cell, or a synthetic polymer.
 7. A pharmaceutical composition comprising a compound according to claim 6 and a pharmaceutical excipient.
 8. A composition comprising a compound according to claim 6 and a diluent.
 9. The process of claim 1 wherein the substrate is selected from the group consisting of a peptide, a protein, a drug, a drug delivery system, a liposome, a virus, a cell, and a synthetic polymer.
 10. The process of claim 9 wherein the substrate is a polypeptide or protein.
 11. The process of claim 1 wherein the starting material is polysialic acid.
 12. The compound of claim 6 wherein R¹ is a polypeptide or protein.
 13. The compound of claim 6 wherein R¹ is a peptide or protein therapeutic. 